(1) We completed analyses of detergent-resistant membrane and lipid profiles of human erythrocytes containing hemoglobin C, a mutant hemoglobin known for to protect children against severe forms of falciparum malaria in West Africa. The manuscript on this work is ready for submission. (2) We also completed the assessment of possible nitric oxide (NO) production in intra-erythrocytic stages of P. falciparum. This work has been published in Journal of Experimental Parasitology. (3) We also have expanded our biophysical/biochemical studies of parasitized erythrocytes including characterization of Zeta potentials associated with the host erythrocyte surface in malaria infection. (4) In this fiscal year, we have started developing a new microscopic technology, termed Total Internal Reflection Microscopy (TIRFM), and combine with novel fluorescence florophore, Tetracysteine. These technologies will be used for Single Molecule Tracking to study dynamic molecular diffusions and interactions of malaria parasite-derived proteins on human erythrocyte membrane. This project will be augmented by cutting-edge imaging technologies, such as Fluorescence Lifetime Microscopy and Quantum Dots. [unreadable] [unreadable] [unreadable] (1) A major transport molecule of human erythrocytes (Band 3) shows clustering in the membrane of human erythrocytes homozygous for hemoglobin C (CC erythrocytes). Erythrocytes that are normal and are homozygous for human hemoglobin (AA) show no such clustering. Our studies have also shown that CC erythrocytes have a clear difference in surface Zeta potential relative to AA erythrocytes. This difference may be a consequence of factors including: (a) hemichrome and serum protein binding to the erythrocyte membrane; (b) protein clustering partially induced by hemichrome aggregation; and/or (c) lipid modification due to hemoglobin denaturation and induced oxidation effects. By examining detergent-resistant fractions of membranes, we found that the distribution of molecules including flotillin-1 (a raft marker), band 3 and CD47 are shifted to denser fractions of CC erythrocytes separated by centrifugation. In contrast, a major structural protein of human erythrocytes (spectrin) was not detected in raft fractions from either AA or CC erythrocytes. [unreadable] [unreadable] We also showed that the lipid compositions of CC and AA erythrocytes carry characteristically different levels of phosphatidylinositol, and phosphatidylserine, all of which are major components of the erythrocyte membranes inner leaflet. Slight changes in lipid composition modulate phase behavior and domain formation in cellular membranes, resulting in a variety of effects on membrane protein clustering and signal transduction. Flowcytometry showed that hemoglobin C erythrocytes display a higher level of phsopatidylserine which helps for modification of outer leaflet molecular distributions. [unreadable] [unreadable] Using model system with hemoglobin A erythrocytes and a strong oxidizing agent, NaNO2, which induce a higher level of hemichrome, we showed that hemichrome level and Zeta poteintial of hemoglobin A erythrocytes in this model system is similar to those in hemoglobin C erythrocytes. These data suggest that hemichrome formation is a major factor to induce hemoglobin C chacteristics and observed band 3 clusterings and modified PfEMP1 display in P. falciparum-infected erythrocytes. [unreadable] [unreadable] (3) In 2006 we initiated studies investigating the possible production of NO in asexual stages of the P. falciparum parasite. In FY2007-2008, we made an important progress in this project, confirming NO is produced in asexual (trophozoites) and sexual (gametocyte) stages of P. falciparum parasites. We confirmed that P. falciparum parasites do not depend on L-arginine, classic substrate for NO production in eukaryotic cells, for the source of NO. Instead, a nitrate reductase mechanism is likely to be responsible for nitrogen incorporation and synthesis of nitric oxide in this organism. Using the P. falciparum genomic database, we identified two possible gene candidates that could be responsible for NO synthetic activity in asexual stages of P. falciparum parasites. We confirmed their expressions in P. falciparum cultures by RT-PCR and generated specific antibodies by DNA vaccine technology. Using specific mice antibodies generated in house against PF13_0353 we obtained signals localized in the food vacuole of trophozoite stage P. falciparum parasites. Our findings are described in a recent publication: gPlasmodium falciparum: Food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite.h Graciela Ostera, Fuyuki Tokumasu, Fabiano Oliveira, Juliana Sa, Tetsuya Furuya, Clarissa Teixeira and James Dvorak. Experimental Parasitology. Vol 120, Issue 1, September 2008, 29-38.[unreadable] [unreadable] This is the first study describing nitric oxide production and function in asexual forms of the P. falciparum parasite. Our initial data indicating a possible NO generation site at the food vacuole, a parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action, could have considerable impact in our understanding of these critical parasitic functions.[unreadable] [unreadable] [unreadable] (3) Zeta potential is a measure of cell-surface electrochemical potential. Preliminary data indicate that this potential changes at the surface of the erythrocyte during parasite growth, and that these changes are associated with the deposition of knob-associated proteins and cytoadherence properties of the infected cell: knobless cell line of P. falciparum has lower zeta potential than that of knob expressing cell lines. The cytoadherence assays showed that cytoadherence for knobless parasite lines are abolished, but slightly recovered by removing sialic acid using neuraminidase. Increase in adherence for knob-presenting erythrocytes was also observed. These data indicate that cytoadherence of parasitized erythrocytes to endothelial cells require knobs, but intercellular adhesion was achieved not only by the intermolecular bindings but also non-specific electrostatic interactions.[unreadable] [unreadable] (4) In this fiscal year, we start developing single-molecule tracking technology for P. falciparum-infected erythrocytes to monitor intermolecular interactions in real-time on live cells. This is a challenging project and never done in other laboratories. In this study, we use etetracysteinef tag which only has four cysteine residues but produces reasonably strong fluorescence when it is combined with a biarsenical labeling reagent. Since it is very small tag, much smaller than GFP, we expect that more natural protein trafficking pattern would be monitored. This tag has never used in malaria study before. This single molecule tracking study will also be augmented by quantum dot (QD)-based single molecule technology. This technology can be used for counter-labeling of erythrocytes. For example, using QD-labeled CD47 in live erythrocytes, we showed that the diffusion coefficient of CD47 has two subpopulations: immobile and mobile pools. We found that P. falciparum infection reduced significantly the mobile pool of CD47, showing that P. falciparum infection dramatically modifies the erythrocyte membrane protein mobility. Our preliminary data using cutting-edge FLIM also revealed this modification. We will combine tetracystein, QDs, FLIM, and custom made TIRFM to perform simultaneous multi-protein tracking to study dynamic intermolecular interactions in real time.